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Plants are the primary source of reusable energy and methods for enhancing the production potential of a given plant are highly sought after. Starch is the major carbohydrate reserve in plants and also the major energy-providing component in human diets. The importance of starch functionality on end product quality, for example in foods, has recently gained increased recognition. Starch textural properties are also important in industrial (non-food) applications where the starch is used as a gelling agent, bulking agent, water retention agent or adhesive, for example.
In cereals, starch makes up approximately 45-65% of the weight of the mature grain. Starch is composed only of glucosidic residues but is found as two types of molecules, amylose and amylopectin, which can be distinguished on the basis of molecular size or other properties. Amylose molecules are essentially linear polymers composed of α-1,4 linked glucosidic units, while amylopectin is a highly branched molecule with α-1,6 glucosidic bonds linking many linear chains of α-1,4 linked glucosidic units. Amylopectin is made of large molecules ranging in size between several tens of thousands to hundreds of thousands of glucose units with around 5 percent α-1,6 branches. Amylose on the other hand is composed of molecules ranging in size between several hundreds to several thousand glucosidic residues with less than one percent branches (for review see Buleon et al., 1998). Wild-type cereal starches typically contain 20-30% amylose while the remainder is amylopectin.
Starch is initially synthesized in plants in chloroplasts of photosynthesizing tissues such as leaves in the form of transitory starch. This is mobilized during subsequent dark periods to supply carbon for export to sink organs and energy metabolism, or for storage in organs such as seeds or tubers. Synthesis and long-term storage of starch occurs in the amyloplasts of the storage organs, where the starch is deposited as semicrystalline granules up to 100 μm in diameter. Granules contain both amylose and amylopectin, the former typically as amorphous material in the native starch granule while the latter is semicrystalline through stacking of the linear glucosidic chains.
The synthesis of starch in the endosperm of higher plants is carried out by a suite of enzymes that catalyse four key steps. Firstly, ADP-glucose pyrophosphorylase activates the monomer precursor of starch through the synthesis of ADP-glucose from G-1-P and adenosine triphosphate (ATP). Secondly, the activated glucosyl donor, ADP-glucose, is transferred to the non-reducing end of a pre-existing α-1,4 linkage by starch synthases. Thirdly, starch branching enzymes introduce branch points through the cleavage of a region of α-1,4 linked glucan followed by transfer of the cleaved chain to an acceptor chain, forming a new α-1,6 linkage. Starch branching enzymes are the only enzymes that can introduce the α-1,6 linkages into α-polyglucans and therefore play an essential role in the formation of amylopectin. Finally, starch debranching enzymes remove some of the branch linkages although the mechanism through which they act is unresolved.
While it is clear that at least these four activities are required for normal starch granule synthesis in higher plants, multiple isoforms of each of the four activities are found in the endosperm of higher plants and specific roles have been proposed for individual isoforms on the basis of mutational analysis or through the modification of gene expression levels using transgenic approaches (Abel et al., 1996, Jobling et al., 1999, Schwall et al., 2000). However, the precise contributions of each isoform of each activity to starch biosynthesis are still not known, and these contributions may differ markedly between species. In the cereal endosperm, two isoforms of ADP-glucose pyrophosphorylase are present, one form within the amyloplast, and one form in the cytoplasm (Denyer et al., 1996, Thorbjornsen et al., 1996). Four classes of starch synthase are found in the cereal endosperm, an isoform exclusively localized within the starch granule, granule-bound starch synthase (GBSS) which is essential for amylose synthesis, two forms that are partitioned between the granule and the soluble fraction (SSI, Li et al., 1999a, SSII, Li et al., 1999b) and a fourth form that is entirely located in the soluble fraction, SSIII (Cao et al, 2000, Li et al., 1999b, Li et al, 2000). Mutations in SSII and SSIII have been shown to alter amylopectin structure (Gao et al, 1998, Craig et al., 1998). No mutations defining a role for SSI activity have been described.
Three forms of branching enzyme are expressed in the cereal endosperm, branching enzyme I (SBEI), branching enzyme IIa (SBEIIa) and branching enzyme IIb (SBEIIb) (Hedman and Boyer, 1982, Boyer and Preiss, 1978, Mizuno et al., 1992, Sun et al., 1997). Genomic and cDNA sequences have been characterized for rice (Nakamura and Yamanouchi, 1992), maize (Baba et al., 1991; Fisher et al., 1993; Gao et al., 1997) and wheat (Repellin et al., 1997; Nair et al., 1997; Rahman et al., 1997). Sequence alignment reveals a high degree of sequence similarity at both the nucleotide and amino acid levels and allows the grouping into the SBEI, SBEIIa and SBEIIb classes. SBEIIa and SBEIIb generally exhibit around 80% sequence identity to each other, particularly in the central regions of the genes.
Two types of debranching enzymes are present in higher plants and are defined on the basis of their substrate specificities, isoamylase type debranching enzymes, and pullulanase type debranching enzymes (Myers et al., 2000). Sugary-1 mutations in maize and rice are associated with deficiency of both debranching enzymes (James et al., 1995, Kubo et al., 1999) however the causal mutation maps to the same location as the isoamylase-type debranching enzyme gene.
Starches extracted from almost all plant species are phosphorylated to some extent. The extent of phosphorylation is usually in the range of 0.1-0.4% of the glucosidic residues, which are phosphorylated at the carbone 3 or the carbone 6 of glucosyl units as phosphate monoesters (Blennow et al, 2000a). Typically, about 80% of the phosphate groups are bound at the C-6 positions, and about 20% at C-3. However, the degree of phosphorylation varies considerably with the botanical source. Starch from potato tuber displays an average of 25 nmoles of glucose-6-phosphate per mg starch while cereal starches display only 1/10th of this amount of glucose-6-phosphate in reserve starch. The presence of phosphate groups in starch affects the water absorption capacity of starch pastes after gelatinization and viscosity properties.
Starch phosphorylation is catalyzed by a group of enzymes belonging to the dikinase family. Two enzymes that carry out starch phosphorylation have been identified in potato and Arabidopsis, namely α-Glucan, Water-Dikinase (GWD; EC 2.7.9.4, otherwise known as the R1 protein or OK1), and Phosphoglucan, Water Dikinase (PWD; EC 2.7.9.5). The former catalyses the transfer of the β-phosphate of ATP to either the C-3 or C-6 position of the glucosyl residue and the γ-phosphate to a water molecule, releasing orthophosphate, while the latter catalyses transfer of phosphates to phosphoglucan (already phosphorylated by GWD) and to water (Baunsgaard et al., 2005; Kotting et al., 2005). More recently Ritte et al. suggested that the phosphorylation in position 3 or 6 of glucosyl residues in starch is catalyzed by the PWD and the GWD respectively (Ritte G. et al., 2006).
Antisense repression of a gene encoding GWD in potatoes reduced starch bound phosphate content by 80% (Viksø-Nielsen et al., 2001). Furthermore, a mutation in a gene designated Sex1 (Starch Excess phenotype) in Arabidopsis thaliana abolished starch phosphorylation, confirming the involvement of GWD as the enzyme responsible (Zeeman and Rees, 1999). In addition both the Arabidopsis mutant and the transgenic antisense potatoes displayed a starch excess phenotype in the leaves, demonstrating a role of GWD in the degradation of transitory starch. Aside from the suppression of starch phosphorylation, no modification of the starch structure was observed in those plants. However, the GWD antisense potatoes showed a reduction in the “cold sweetening” phenotype in tubers as well as the starch excess phenotype in the leaves (Lorberth et al., 1998). The potato plants also showed an increase in tuber number associated with a decrease of individual tuber weight, but did not show any other effect on starch accumulation in the tuber. The Arabidopsis sex1 mutants which were affected in their transitory starch metabolism also had altered carbohydrate metabolism, grew slowly and flowered late (Yu et al., 2001).
The relationship between starch degradation and starch phosphate content remains unclear. The starch produced by the antisense lines from potato displayed a high resistance to β-amylase degradation, suggesting that starch phosphorylation may be a prerequisite for degradation by β-amylase. Phosphorylated residues could be a targeting signal for this enzyme in order to degrade starch during the night period. Some studies have suggested an association of α-amylase and the R1 (GWD) protein with the starch granule before the degradation initiated.
Starch degradation and phosphorylation in germinating cereal seeds such as wheat is less understood and is a highly specialized system involving tissue deterioration and induction of hydrolytic enzymes as well as starch degradation.
Wheat is a staple food in many countries and supplies approximately 20% of the food kilojoules for the total world population. The processing characteristics of wheat make it the preferred base for most cereal-based processed products such as bread, pasta and noodles. Wheat consumption is increasing world-wide with increasing affluence. Breadwheat (Triticum aestivum) is a hexaploid having three different genomes, A, B and D, and most of the known genes in wheat are present in triplicate, one on each genome. The hexaploid nature of the breadwheat genome makes finding and combining gene mutations in each of the three genomes a challenge. The presence of three genomes has a buffering effect by masking mutations in individual genomes, in contrast to the more readily identified mutations in diploid species. Known variation in wheat starch structure has been limited relative to the variation available in maize or rice. Another contributing factor to this is that the transformation efficiency of wheat has lagged behind that for other cereals. It is believed that genes involved in starch phosphorylation in wheat and their effects have not been studied previously, and it is unknown whether effects observed on starch phosphorylation in potato and Arabidopsis, both dicots, could be similarly replicated in monocot species such as wheat.